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Interleukin-10 inhibits HIV-1 LTR-directed gene expression in human macrophages through the induction of cyclin T1 proteolysis
Virology 352 (2006) 485 – 492 www.elsevier.com/locate/yviro
Interleukin-10 inhibits HIV-1 LTR-directed gene expression in human macrophages through the induction of cyclin T1 proteolysis Yan Wang, Andrew P. Rice ⁎ Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, One Baylor Plaza, Houston, TX 77030, USA Received 17 April 2006; returned to author for revision 5 May 2006; accepted 11 May 2006 Available online 15 June 2006
Abstract Regulation of HIV-1 replication in human monocytes/macrophages occurs at multiple levels including transcription of the proviral genome, which depends on virally encoded Tat protein. Interleukin-10 (IL-10), an anti-inflammatory cytokine which is up-regulated during disease progression of AIDS, has been reported to suppress HIV-1 replication in macrophages at a post-entry stage of the virus life cycle. Our previous studies have demonstrated that Tat function is regulated during the differentiation of monocyte-derived macrophages (MDMs) in a manner that correlates with the early induction and subsequent shut-off of its cellular cofactor cyclin T1. Here, we report that IL-10 down-regulates cyclin T1 expression through the induction of proteasome-mediated proteolysis in human macrophages. Using a reporter virus that is deficient in Tat function, we also demonstrate that IL-10 inhibits HIV-1 gene expression in a Tat-dependent manner. Together, these results suggest that the downregulation of cyclin T1, and consequently Tat function, contributes to the suppressive effect of IL-10 on HIV-1 replication in human macrophages. © 2006 Elsevier Inc. All rights reserved. Keywords: HIV; Tat; IL-10; Cyclin T1; P-TEFb; Transcription; Proteolysis
Introduction Cells from the monocyte/macrophage lineage are one of the major targets for human immunodeficiency virus (HIV-1) infection due to the expression of CD4 and CCR5 molecules on their cell surface. Although infection appears to be generally less productive than that in CD4+ T lymphocytes, monocytes and macrophages are believed to make important contributions to the transmission and pathogenesis of HIV-1 infection throughout the course of disease progression (Verani et al., 2005; Kedzierska and Crowe, 2002; Swingler et al., 1999), especially at the late stages of acquired immunodeficiency syndrome (AIDS) when CD4+ T lymphocytes have been extensively depleted (Orenstein et al., 1997). Several different steps of HIV-1 replication have been reported to be regulated in monocytes/macrophages, including virus entry, nuclear import, transcription of the provirus, and virus assembly/release (Verani et al., 2005).
⁎ Corresponding author. Fax: +1 713 798 3490. E-mail address: [email protected] (A.P. Rice). 0042-6822/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2006.05.013
Efficient transcriptional elongation of the HIV-1 proviral genome by cellular RNA polymerase II (RNAP II) requires the virus-encoded transactivator protein known as Tat (Karn, 1999; Garber and Jones, 1999; Rice and Herrmann, 2003; Barboric and Peterlin, 2005). Tat activates transcription by recruiting a cellular kinase complex composed of Cdk9 and cyclin T1 to the TAR RNA stem-loop structure, located at the 5′ end of the nascent RNA transcript. The Cdk9 and cyclin T1 complex is one of the several kinase complexes expressed in human cells that are collectively termed P-TEFb. Each P-TEFb complex contains the catalytic subunit Cdk9, expressed as both a major 42-kDa and a minor 55-kDa isoform, plus a regulatory subunit, either cyclin T1, cyclin T2, or cyclin K (Liu and Herrmann, 2005; Price, 2000; Shore et al., 2003). Tat binds directly to cyclin T1 and therefore specifically recruits the cyclin T1containing P-TEFb complex. Upon the formation of the TARTat-P-TEFb ternary complex, transcriptional elongation is activated by Cdk9 through hyperphosphorylation of the carboxyl-terminal domain (CTD) of RNAP II and phosphorylation of negative factors that associate with RNAP II and limit elongation (Price, 2000; Ivanov et al., 2000; Kim and Sharp, 2001; Garber and Jones, 1999; Fujinaga et al., 2004).
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Our previous studies have demonstrated that the cellular levels of cyclin T1 protein are transiently induced by posttranscriptional mechanisms during the differentiation of monocytes toward macrophages in vitro (Liou et al., 2002). Freshly isolated monocytes contain high levels of cyclin T1 mRNA but express very low amount of cyclin T1 protein. During the first few days of differentiation, cyclin T1 protein expression is upregulated without an increase in mRNA levels. After 7 to 10 days, however, cyclin T1 expression is shut-off by a posttranscriptional mechanism involving proteasome-mediated proteolysis (Liou et al., 2004). Plasmid transfection assays in monocyte-derived macrophages (MDM) show parallel regulation of Tat transactivation activity, suggesting that cyclin T1 is a limiting factor for Tat function in MDMs (Liou et al., 2002). Interestingly, HIV-1 infection has also been shown to prevent the shut-off of cyclin T1 protein expression in macrophages (Liou et al., 2004). Cytokines and chemokines are host factors that continuously regulate HIV-1 infection and replication in cells of the myeloid lineage (Kedzierska et al., 2003). IL-10 is a potent immuno-
suppressive cytokine which inhibits macrophage activation by decreasing the production of pro-inflammatory cytokines such as IL-6 and TNF-α (Moore et al., 2001). Elevated serum levels of IL-10 and IL-4, concomitant with loss of IL-2 and IFN-γ production, have been suggested to mark the progression to AIDS (Clerici and Shearer, 1993). Since cells from the monocytes/macrophages lineage play a pivotal role in HIV-1 persistence and pathogenesis, especially in AIDS patients whose CD4+ lymphocytes have been extensively depleted (Verani et al., 2005; Orenstein et al., 1997), it is important to understand the interactions between IL-10 and the HIV-1 replication in the specific setting of monocytes/macrophages. Previously, IL-10 has been reported to suppress HIV-1 replication in monocytes/macrophages at early stages of infection, although virus entry does not appear to be affected (Naif et al., 1996; Ancuta et al., 2001; Kootstra et al., 1994; Wang et al., 1998). However, mechanisms of suppression by IL10 have not yet been elucidated. In this study, we used primary human peripheral blood MDMs to investigate the effects of IL-10 on cyclin T1
Fig. 1. IL-10 inhibits cyclin T1 expression during MDM differentiation. (A) Flow cytometric analysis of cell surface markers. Day 0 monocytes and day 4 macrophages were stained with anti-CD14 or anti-CD71 antibodies (open area). Unstained cells were used as controls (shaded area). (B) 20 μg of total cell lysates prepared at indicated time points were examined by SDS-PAGE followed with immunoblot using the indicated antisera. β-Actin was used as a loading control. (C) Cells were lysed at 6, 14, and 24 h after the addition of IL-10 and were analyzed for expression of the indicated proteins by immunoblotting. For all experiments, a final concentration of 10 ng/ml of IL-10 was used.
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expression and HIV-1 replication. We found that IL-10 downregulates cellular levels of cyclin T1 protein through the induction of proteasome-mediated proteolysis. Furthermore, we found that IL-10 suppresses HIV-1 gene expression in infected MDMs by inhibiting Tat function. Results IL-10 represses cyclin T1 protein expression in differentiating macrophages To examine the effects of IL-10 on cyclin T1 expression, MDMs were differentiated from monocytes using GM-CSF. Flow cytometric analysis showed decreased expression of monocytic marker CD14 and increased expression of macrophage marker CD71 in cells that were not treated with IL-10, indicating the expected differentiation of MDMs (Fig. 1A). MDM cultures treated from day 2 of in vitro differentiation with 10 ng/ml of IL-10 in addition to GM-CSF demonstrated sustained expression of CD14 on day 4. The induction of CD71 surface expression on these cells is similar to that of the MDMs differentiated without IL-10, suggesting that MDMs undergo similar maturation in the presence of IL10 (Fig. 1A). Interestingly, cyclin T1 protein expression was greatly reduced after cells were differentiated in the presence of IL-10 for 2 days (Fig. 1B, compare lanes 2 and 3), while the expression of the 42-kDa isoform of Cdk9, which forms a P-TEFb complex together with cyclin T1, was largely unaffected at this time. To further characterize the kinetics of this down-regulation, cell lysates were prepared at 6, 14, or
Fig. 3. IL-10 down-regulates cyclin T1 expression in macrophages by inducing proteasome-mediated proteolysis. The proteasome inhibitor MG101 (50 nM) was added to IL-10-treated macrophages for 1 h before cells were lysed. 20 μg of total lysates was separated by SDS-PAGE. Levels of indicated proteins were examined by immunoblotting. Data from two independent experiments are presented here.
24 h after the addition of IL-10 on day 2, and the expression levels of cyclin T1 were examined by immunoblot (Fig. 1C). Down-regulation of cyclin T1 was not observed at 6 h after IL-10 addition. It became apparent by 14 h and by 24 h that cyclin T1 was nearly undetectable. These results indicate that the effect of IL-10 on cyclin T1 is a relatively slow process and may be a late event induced by the cytokine. We observed similar kinetics of cyclin T1 down-regulation by IL10 in MDMs from a number of other blood donors. We therefore conclude that IL-10 down-regulates cyclin T1 expression in human macrophages by a relatively slow process, which is likely to be a downstream effect of IL-10 binding to its receptor. IL-10 does not affect cyclin T1 RNA expression
Fig. 2. IL-10 does not affect cyclin T1 RNA expression. Total RNA was isolated from macrophages treated with or without 10 ng/ml of IL-10 at the indicated time points. RNAs were examined by real-time RT-PCR as previously described (Haaland et al., 2005) for the detection of cyclin T1 and CD11c cDNAs. Abundance of the transcripts was normalized to that of GAPDH. Inhibition of cyclin T1 by IL-10 at the protein level was confirmed by immunoblot (see Fig. 1B).
To explore the underlying mechanisms of cyclin T1 downregulation by IL-10, we examined cyclin T1 mRNA levels by quantitative real-time RT-PCR (Fig. 2). As we previously reported using cells differentiated with GM-CSF alone (Liou et al., 2002), cyclin T1 mRNA levels are significantly higher in monocytes than MDMs differentiated for 4 or 7 days. This contrasts to the near absence of cyclin T1 protein expression in day 0 monocytes, and the high-level protein expression in day 4 MDMs (Fig. 1B). Although IL-10 was able to dramatically decrease the level of cyclin T1 protein in day 4 MDMs (Fig. 1B), the amount of cyclin T1 mRNA remained similar in MDMs treated with and without IL-10 (Fig. 2). As a control to validate our real-time RT-PCR assay, RNA levels of CD11c were also examined. CD11c is an integrin whose protein expression is known to be up-regulated through a transcriptional mechanism (Noti and Reinemann, 1995). As expected, CD11c mRNA was strongly induced from day 0 to day 4 in cells treated with GM-CSF alone, while the addition of IL-10 had only a small negative effect on cyclin T1 mRNA on day 4. We therefore conclude that the down-regulation of cyclin T1 expression by IL-10 is not the result of decreased levels of cyclin T1 mRNA.
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ated protein degradation. This process is accelerated by IL-10, and it appears to be the major mechanism involved in the inhibition of cyclin T1 expression by IL-10. IL-10 inhibits cyclin T1 expression in HIV-1-infected macrophages
Fig. 4. IL-10 inhibits cyclin T1 protein expression in HIV-1-infected macrophages. MDMs were infected on day 2 with NL4-3-Luc virus. 10 ng/ml of IL-10 was added to culture on day 3. Cell lysates were prepared on indicated time points and examined by immunoblot.
IL-10 down-regulates cyclin T1 expression through proteasome-mediated proteolysis Because MDMs differentiated with GM-CSF down-regulate cyclin T1 expression at a late differentiation stage by a proteasome-mediated process (Liou et al., 2002), we were interested in examining whether IL-10 activates the same process. We blocked proteasome-mediated proteolysis in IL-10treated macrophages with the proteasome inhibitor, MG101. As shown in Fig. 3 for MDMs from 2 donors, treatment with MG101 for 1 h prior to preparation of cell extracts was able to overcome the suppressive effect of IL-10 on cyclin T1 expression. Treatment with MG101 had no significant effect on the expression of CD11c, the 42-kDa Cdk9, or β-actin, indicating the specificity of its effect on cyclin T1. These results, together with our previous findings (Liou et al., 2002, 2004), indicate that the down-regulation of cyclin T1 protein in differentiating macrophages is mediated by proteasome-medi-
Our previous studies have demonstrated induction of cyclin T1 protein expression by HIV-1 infection (Liou et al., 2004). We therefore examined whether this induction can be overridden by the effect of IL-10. MDMs were infected on day 2 with NL4-3Luc, a reporter virus that encodes the full-length HIV-1 proviral DNA and expresses luciferase enzyme in the place of Nef (Connor et al., 1995). Consistent with our previous findings, cyclin T1 protein expression was shut-off at day 5 in uninfected cultures, whereas it was sustained in the infected culture that was not treated with IL-10 (Fig. 4). In contrast, cyclin T1 expression was absent at both days 4 and 5 in infected cultures that were treated with IL-10. These results indicate that under these conditions, the repression of cyclin T1 expression by IL10 is dominant over the induction by HIV-1 infection. Suppression of cyclin T1 expression by IL-10 contributes to its inhibition on HIV-1 replication in human macrophages Previous studies have indicated that IL-10 specifically inhibits HIV-1 replication in primary human MDMs at a post-entry stage (Ancuta et al., 2001; Kootstra et al., 1994; Wang et al., 1998; Naif et al., 1996). We next examined whether the down-regulation of cyclin T1 by IL-10 contributes to the inhibition of HIV-1 replication in MDMs. To investigate the impact of IL-10 on Tat-mediated
Fig. 5. IL-10 inhibits HIV-1 gene expression in macrophages by suppressing Tat function. (A) 293T cells were infected with either NL4-3-Luc or NL4-3-Luc-Tat− virus. Expression of wild-type Tat protein or mutant Tat-pro18IS was achieved by transfection (empty expression vector was used as a control). Cell lysates were prepared 48 h post-transfection and analyzed for luciferase activity. (B) Differentiating MDMs were infected in triplicate on day 1 with either NL4-3-Luc or NL4-3Luc-Tat− by spinoculation (O'Doherty et al., 2000b). IL-10 (10 ng/ml) was added into culture media on day 3. Cells were harvested every day beginning at day 5. Suppression of cyclin T1 expression in IL-10-treated macrophages was confirmed by immunoblotting (data not shown). Luciferase activities were examined as described above.
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transcription, we constructed a mutant HIV-1 reporter virus, NL4-3Luc-Tat− that lacks a functional Tat protein. This Tat mutant, Tatpro18IS, has been shown previously by plasmid transfection assays to abolish Tat transactivation (Rice and Carlotti, 1990). In infections using 293T cells, the wild-type reporter virus, NL4-3-Luc, expresses efficiently, while mutation of Tat in the NL4-3-LucTat− virus reduced viral expression by ∼32-fold (Fig. 5A). Luciferase expression of the NL4-3-Luc-Tat− virus could be restored to a similar level as that of the wild-type virus by cotransfecting a plasmid expressing the wild-type Tat but not a plasmid expressing the mutant Tat-pro18IS. These results indicate that NL4-3-Luc-Tat− is defective in Tat function, yet it can be fully complemented by exogenously expressing wild-type Tat. We then tested the effects of IL-10 on MDMs infected with these two viruses. MDMs were infected with either NL4-3-Luc or NL4-3Luc-Tat− on day 1 and treated with IL-10 on day 3. From day 5 to day 7, luciferase expression in NL4-3-Luc-infected MDMs was less in IL-10-treated cells than that in untreated cells (Fig. 5B). In contrast, luciferase activities in MDMs infected with the mutant virus, NL4-3-Luc-Tat-, were similar in cells treated with or without IL-10, although the overall expression level in the Tat− virusinfected cells was ∼30-fold lower than the wild-type virus due to the absence of Tat transactivation (Fig. 5B). We observed similar results in infections with these viruses in MDMs from several blood donors. These results indicate that the inhibition of Tat transactivation by IL-10 contributes to the suppression of HIV-1 gene expression by this cytokine. Discussion Despite the well-documented anti-inflammatory functions of IL10, the mechanisms through which IL-10 suppresses macrophage activation are diverse and poorly understood. Phosphorylation and activation of the Jak/Stat pathway are thus far the best-characterized signaling event triggered by IL-10 in macrophages. However, previous studies indicate that one or more additional pathways must also be activated to achieve the inhibitory effect of IL-10 on macrophage activation (Moore et al., 2001). The results presented here show that the IL-10 is able to down-regulate the expression of cyclin T1, a major component of the P-TEFb transcription elongation complex in human MDMs. The suppression of cyclin T1 by IL-10 appears to be due to the induction of proteasomemediated proteolysis, which, to our knowledge, is a novel observation on cellular events triggered by IL-10. It is not clear, however, what signal(s) causes the induction of degradation in MDMs differentiated with GM-CSF in the absence of IL-10. We believe that it is unlikely that the accumulation of IL-10 in the culture medium triggers this event, as we have been unable to detect any activity in the conditioned culture medium from day 7 MDMs, where cyclin T1 had been expressed at low levels, which accelerated the down-regulation of cyclin T1 in early MDM cultures (unpublished results). We also attempted to block the effect of IL-10 by neutralizing it with monoclonal antibodies. We were able to block the down-regulation of cyclin T1 by mixing exogenous IL-10 with antibodies before cytokine addition to cells. However, we have been unable to observe any effect on cyclin T1 expression after the addition of antibodies against any
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endogenous IL-10 which may be secreted into culture media during macrophage differentiation (unpublished data). If IL-10 is involved in the natural shut-off of cyclin T1 expression that occurs late in differentiation in vitro, it is possible that its effect is carried out through autocrine functions rather than paracrine signaling. Proteasome-mediated protein degradation has been identified as a regulatory mechanism for many cellular processes (Pickart, 2004; Ciechanover, 1994). Proteins that undergo rapid turnover usually contain sequence identifiers for targeted degradation, such as PEST regions, KEFRQ motifs and cyclin destruction boxes (Rechsteiner and Rogers, 1996). Cyclin T1 contains a putative PEST sequence in its carboxyl terminus (Wei et al., 1998), which may specify cyclin T1 degradation by the ubiquitylation and proteasome pathway. Alternatively, it is possible that an unidentified cellular factor is required for the translation of cyclin T1 mRNA, and this positive factor may be down-regulated through IL-10-induced proteolysis; blockage of proteasome-mediated proteolysis by MG101 may increase the level of this putative positive factor, and this would result in increased translation of cyclin T1 mRNA and a subsequent increase in the level of cyclin T1 protein. Our data cannot distinguish these possibilities, although we favor the former because it is commonly involved in regulating levels of cellular transcription factors and cyclin-cdk complexes (Obaya and Sedivy, 2002; Muratani and Tansey, 2003). Interestingly, suppressing cyclin T1 expression through proteasome-mediated degradation appears to be specific for macrophages as treatment of peripheral blood lymphocytes, Jurkat, and 293T cells with proteasome inhibitors does not affect its protein level (Liou et al., 2004). Transcription profiling studies in control MDMs and MDMs treated with IL-10 are currently underway in an attempt to identify enzymes and accessory proteins induced by IL-10 that may be involved in the down-regulation of cyclin T1. To date, reports on the impact of IL-10 on HIV-1 replication in monocytes/macrophages have been divergent and dependent on the cell types and amounts of cytokine used in the experiments (Stearns et al., 2003; Sozzani et al., 1998; Ancuta et al., 2001; Kootstra et al., 1994; Naif et al., 1996; Wang et al., 1998). Our data are also consistent with the findings that IL-10, by itself, affects HIV-1 replication negatively in macrophages. In conclusion, our results demonstrate that IL-10 downregulates the expression of cyclin T1, probably though the activation of proteasome-mediated proteolysis of this protein. We also present evidence that the inhibition of HIV-1 replication in MDMs by IL-10 involves the suppression of Tat transactivation function during the transcription of HIV-1 provirus. Our findings, together with other reports (Moore et al., 2001), suggest that IL-10 may affect multiple stages of the HIV-1 life cycle, and its induced expression in progressive AIDS patients may have a complex effect on HIV-1 pathogenesis. Materials and methods Reagents and antibodies Human granulocyte–macrophage colony-stimulating factor (GM-CSF) and interleukin-10 (IL-10) were purchased from R&D
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Systems. Anti-CD14-PE and anti-CD71-FITC were from BD PharMingen. Primary antibodies against cyclin T1 (T-18), Cdk9 (C-20) and CD11c (C-20) were purchased from Santa Cruz Biotechnology. Anti-β-actin antibody and MG101 were from Sigma. Isolation and differentiation of monocytes/macrophages Peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coat of healthy blood donors (Gulf Coast Regional Blood Center) by Ficoll-Hypaque density gradient centrifugation (Isolymph [Gallard/Schlesinger]). Monocytes were isolated by an adherence method in which 3 × 107 PBMCs were seeded onto each 10-cm plastic tissue culture dish (Sarstedt) containing RPMI-1640 (Invitrogen) supplemented with 1% human serum (Sigma) and 1% antibiotics (Invitrogen). Following incubation at 37 °C for 1 h, non-adherent cells were removed, and adherent monocytes were washed vigorously with pre-warmed phosphate-buffered saline (PBS). Cells were further purified by incubating at 37 °C for 2 h in complete medium (RPMI-1640 + 10% fetal bovine serum + antibiotics) and washing with pre-warmed PBS vigorously for 3 times. Monocytes were then cultured in complete medium containing 10 U/ml GM-CSF to allow differentiation into macrophages. For IL-10 treatment, a one-time addition of IL-10 was introduced on day 2 to a final concentration of 10 ng/ml. Initial seeding conditions for other formats of tissue culture vessels are 2 × 106 PBMCs per 6-cm dish or 1 × 106 PBMCs per well in a 6-well plate. In some experiments, monocytes were also isolated from PBMCs using the negative selection-based Monocyte Isolation Kit II (Miltenyi Biotech.) to achieve a purity of 90% or higher as determined by flow cytometry.
by measuring OD260 nm of the sample. 1 μg of the total RNA was reverse transcribed using iScript cDNA synthesis kit (BioRad) in a 20 μl reaction according to the manufacturer's protocol. Products from these reactions were diluted 1:10 and used as templates in 15 μl real-time PCR reactions containing 1/ 2 volume of 2× iQ SYBR Green Supermix (Bio-Rad) and 200 nM final concentration of the appropriate primers. Triplicate PCR reactions were run in 96-well format using a Bio-Rad iCycler. After the initial hot start at 95 °C for 3 min, 40 cycles of amplification were carried out for 15 s at 95 °C followed by 1-min annealing/extension at 55 °C (data were collected at this step). Analysis was performed using the MyIQ software program (Bio-Rad), which automatically determines the threshold crossing (CT) value for each reaction. A five-fold dilution series of HeLa cell total RNA was used to determine the amplification efficiency (E) of each primer pair. The relative expression ratio (R) of a gene of interest (g.o.i., normalized to day 0) was calculated with the following formula using GAPDH as a reference gene (Pfaffl, 2001). DCTg:o:i:ðday 0sampleÞ 1 þ Eg:o:i: R¼ ð1 þ Eref ÞDCTref ðday 0sampleÞ Sequences of the primers used here are as follows: cyclin T1 (sense 5′-AACCTTCGCCGCTGCCTTC-3′; anti-sense 5′ACC GTTTGTTGTTGTTCTTCCTCTC-3′), CD11c (sense 5′-TTTGAGTGTCGGGAGCAGGTG-3′; anti-sense 5′GCCAGGTCCAAGGTCACAGAG-3′), GAPDH (sense 5′CGCCAGCCGAGCCACATC-3′; anti-sense 5′-AATCCCTTGACTCCGACCTTCA-3′). Virus production and infections
Flow cytometry Adherent cells were collected by incubating the dishes at 4 °C with PBS containing 2% FBS and 2 mM EDTA for 10 min and detaching the cells using a rubber scraper. Cells were washed twice with cold PBS containing 2% FBS and stained for 15 min at 4 °C with appropriate antibodies. After washing, cells were fixed in 1% paraformaldehyde (Fisher) and analyzed using a Coulter XL-MCL flow cytometer. Immunoblots Cell lysates were prepared using Luciferase Cell Lysis Buffer (Promega) according to the manufacturer's protocol. Protein concentration was determined by a Bradford assay (Bio-Rad). 20 μg of total lysate was separated by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). The procedure for immunoblotting using SuperSignal West Pico Chemiluminescent Substrate (Pierce) was described previously (Herrmann et al., 1996). Isolation of total RNA and analysis by quantitative real-time RT-PCR Total RNA was extracted from frozen cell pellets using the RNeasy mini kit (Qiagen). RNA concentration was determined
pNL-Luc-E−R+ was obtained from the NIH AIDS Research and Reference Reagent Program and has been described previously (Connor et al., 1995). pNL-Luc-E−R+ encodes for full-length NL4-3 HIV-1 proviral DNA with a frameshift in Env and expresses luciferase enzyme in place of Nef. To generate the Tat-null virus, NL4-3-LucTat−, an EcoRI restriction endonuclease site (GAATTC) was inserted between residues 18 and 19 within the Tat-encoding sequence of pNL-Luc-E−R+ using QuickChange II XL site-directed mutagenesis kit (Stratagene). For virus production, 5 × 106 293T cells were transfected with 10 μg of either pNL-Luc-E-R+ or pNL-Luc-E−R+Tat−, along with 10 μg of plasmids expressing VSV-G protein (pseudotyped envelope) and HIV-1 Tat protein by calcium phosphate transfection (Sambrook and Russell, 2001). Culture supernatant containing infectious virions was harvested 72 h posttransfection and cleared of cell debris by centrifuging at 1000×g for 10 min. Virus concentration was determined by the COULTER HIV-1 p24 antigen assay using protocols supplied by the manufacturer. Virus stock was stored at −80 °C in working aliquots. For infection of 293T cells, 3 × 105 293T cells (ATCC) in 12-well plates were infected in triplicate with 5 μg of either NL4-3-Luc or NL4-3-Luc-Tat−. Infection of primary human MDMs was carried out in 6-well plates using 50 μg/ml of either virus. The method of spinoculation was employed to
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achieve efficient infection. As previously described (O'Doherty et al., 2000a), cells were incubated with virus-containing media under 2000×g of centrifugational force for 2 h. Unbound virus was removed by washing the cells 3 times with cold PBS. Complete media containing 10 U/ml GM-CSF were added to the cells for further culturing. Transfections and luciferase assay Transient transfection was carried out using Lipofectamine 2000 transfection reagent (Invitrogen). 3 × 105 293T cells, infected with either NL4-3-Luc or NL4-3-Luc-Tat− , were transfected in triplicates with 0.5 μg of either the control vector, Tat-expressing or Tat mutant-expressing plasmid. Cells were harvested 48 h later. Luciferase activity in cell lysates was detected using the Luciferase Assay System from Promega according to protocols supplied therein. Acknowledgments We thank Dr. Ned Landau for the kind gift of plasmid pNL43.Luc.R+.E−. We also thank Dr. Chris Herrmann for comments on the manuscript and Dr. Li-Ying Liou for useful discussions and technical advice. This work was supported by NIH grant AI45374. References Ancuta, P., Bakri, Y., Chomont, N., Hocini, H., Gabuzda, D., HaeffnerCavaillon, N., 2001. Opposite effects of IL-10 on the ability of dendritic cells and macrophages to replicate primary CXCR4-dependent HIV-1 strains. J. Immunol. 166, 4244–4253. Barboric, M., Peterlin, B.M., 2005. A new paradigm in eukaryotic biology: HIV Tat and the control of transcriptional elongation. PLoS Biol. 3, e76. Ciechanover, A., 1994. The ubiquitin-proteasome proteolytic pathway. Cell 79, 13–21. Clerici, M., Shearer, G.M., 1993. A TH1→TH2 switch is a critical step in the etiology of HIV infection. Immunol. Today 14, 107–111. Connor, R.I., Chen, B.K., Choe, S., Landau, N.R., 1995. Vpr is required for efficient replication of human immunodeficiency virus type-1 in mononuclear phagocytes. Virology 206, 935–944. Fujinaga, K., Irwin, D., Huang, Y., Taube, R., Kurosu, T., Peterlin, B.M., 2004. Dynamics of human immunodeficiency virus transcription: P-TEFb phosphorylates RD and dissociates negative effectors from the transactivation response element. Mol. Cell. Biol. 24, 787–795. Garber, M.E., Jones, K.A., 1999. HIV-1 Tat: coping with negative elongation factors. Curr. Opin. Immunol. 11, 460–465. Haaland, R.E., Yu, W., Rice, A.P., 2005. Identification of LKLF-regulated genes in quiescent CD4+ T lymphocytes. Mol. Immunol. 42, 627–641. Herrmann, C.H., Gold, M.O., Rice, A.P., 1996. Viral transactivators specifically target distinct cellular protein kinases that phosphorylate the RNA polymerase II C-terminal domain. Nucleic Acids Res. 24, 501–508. Ivanov, D., Kwak, Y.T., Guo, J., Gaynor, R.B., 2000. Domains in the SPT5 protein that modulate its transcriptional regulatory properties. Mol. Cell. Biol. 20, 2970–2983. Karn, J., 1999. Tackling Tat. J. Mol. Biol. 293, 235–254. Kedzierska, K., Crowe, S.M., 2002. The role of monocytes and macrophages in the pathogenesis of HIV-1 infection. Curr. Med. Chem. 9, 1893–1903. Kedzierska, K., Crowe, S.M., Turville, S., Cunningham, A.L., 2003. The influence of cytokines, chemokines and their receptors on HIV-1
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human monocytes/macrophages through modulation of CCR5 expression. J. Virol. 72, 7642–7647. Wei, P., Garber, M.E., Fang, S.M., Fischer, W.H., Jones, K.A., 1998. A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA. Cell 92, 451–462.
Interleukin-10 inhibits HIV-1 LTR-directed gene expression in human macrophages through the induction of cyclin T1 proteolysis Yan Wang, Andrew P. Rice ⁎ Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, One Baylor Plaza, Houston, TX 77030, USA Received 17 April 2006; returned to author for revision 5 May 2006; accepted 11 May 2006 Available online 15 June 2006
Abstract Regulation of HIV-1 replication in human monocytes/macrophages occurs at multiple levels including transcription of the proviral genome, which depends on virally encoded Tat protein. Interleukin-10 (IL-10), an anti-inflammatory cytokine which is up-regulated during disease progression of AIDS, has been reported to suppress HIV-1 replication in macrophages at a post-entry stage of the virus life cycle. Our previous studies have demonstrated that Tat function is regulated during the differentiation of monocyte-derived macrophages (MDMs) in a manner that correlates with the early induction and subsequent shut-off of its cellular cofactor cyclin T1. Here, we report that IL-10 down-regulates cyclin T1 expression through the induction of proteasome-mediated proteolysis in human macrophages. Using a reporter virus that is deficient in Tat function, we also demonstrate that IL-10 inhibits HIV-1 gene expression in a Tat-dependent manner. Together, these results suggest that the downregulation of cyclin T1, and consequently Tat function, contributes to the suppressive effect of IL-10 on HIV-1 replication in human macrophages. © 2006 Elsevier Inc. All rights reserved. Keywords: HIV; Tat; IL-10; Cyclin T1; P-TEFb; Transcription; Proteolysis
Introduction Cells from the monocyte/macrophage lineage are one of the major targets for human immunodeficiency virus (HIV-1) infection due to the expression of CD4 and CCR5 molecules on their cell surface. Although infection appears to be generally less productive than that in CD4+ T lymphocytes, monocytes and macrophages are believed to make important contributions to the transmission and pathogenesis of HIV-1 infection throughout the course of disease progression (Verani et al., 2005; Kedzierska and Crowe, 2002; Swingler et al., 1999), especially at the late stages of acquired immunodeficiency syndrome (AIDS) when CD4+ T lymphocytes have been extensively depleted (Orenstein et al., 1997). Several different steps of HIV-1 replication have been reported to be regulated in monocytes/macrophages, including virus entry, nuclear import, transcription of the provirus, and virus assembly/release (Verani et al., 2005).
⁎ Corresponding author. Fax: +1 713 798 3490. E-mail address: [email protected] (A.P. Rice). 0042-6822/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2006.05.013
Efficient transcriptional elongation of the HIV-1 proviral genome by cellular RNA polymerase II (RNAP II) requires the virus-encoded transactivator protein known as Tat (Karn, 1999; Garber and Jones, 1999; Rice and Herrmann, 2003; Barboric and Peterlin, 2005). Tat activates transcription by recruiting a cellular kinase complex composed of Cdk9 and cyclin T1 to the TAR RNA stem-loop structure, located at the 5′ end of the nascent RNA transcript. The Cdk9 and cyclin T1 complex is one of the several kinase complexes expressed in human cells that are collectively termed P-TEFb. Each P-TEFb complex contains the catalytic subunit Cdk9, expressed as both a major 42-kDa and a minor 55-kDa isoform, plus a regulatory subunit, either cyclin T1, cyclin T2, or cyclin K (Liu and Herrmann, 2005; Price, 2000; Shore et al., 2003). Tat binds directly to cyclin T1 and therefore specifically recruits the cyclin T1containing P-TEFb complex. Upon the formation of the TARTat-P-TEFb ternary complex, transcriptional elongation is activated by Cdk9 through hyperphosphorylation of the carboxyl-terminal domain (CTD) of RNAP II and phosphorylation of negative factors that associate with RNAP II and limit elongation (Price, 2000; Ivanov et al., 2000; Kim and Sharp, 2001; Garber and Jones, 1999; Fujinaga et al., 2004).
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Our previous studies have demonstrated that the cellular levels of cyclin T1 protein are transiently induced by posttranscriptional mechanisms during the differentiation of monocytes toward macrophages in vitro (Liou et al., 2002). Freshly isolated monocytes contain high levels of cyclin T1 mRNA but express very low amount of cyclin T1 protein. During the first few days of differentiation, cyclin T1 protein expression is upregulated without an increase in mRNA levels. After 7 to 10 days, however, cyclin T1 expression is shut-off by a posttranscriptional mechanism involving proteasome-mediated proteolysis (Liou et al., 2004). Plasmid transfection assays in monocyte-derived macrophages (MDM) show parallel regulation of Tat transactivation activity, suggesting that cyclin T1 is a limiting factor for Tat function in MDMs (Liou et al., 2002). Interestingly, HIV-1 infection has also been shown to prevent the shut-off of cyclin T1 protein expression in macrophages (Liou et al., 2004). Cytokines and chemokines are host factors that continuously regulate HIV-1 infection and replication in cells of the myeloid lineage (Kedzierska et al., 2003). IL-10 is a potent immuno-
suppressive cytokine which inhibits macrophage activation by decreasing the production of pro-inflammatory cytokines such as IL-6 and TNF-α (Moore et al., 2001). Elevated serum levels of IL-10 and IL-4, concomitant with loss of IL-2 and IFN-γ production, have been suggested to mark the progression to AIDS (Clerici and Shearer, 1993). Since cells from the monocytes/macrophages lineage play a pivotal role in HIV-1 persistence and pathogenesis, especially in AIDS patients whose CD4+ lymphocytes have been extensively depleted (Verani et al., 2005; Orenstein et al., 1997), it is important to understand the interactions between IL-10 and the HIV-1 replication in the specific setting of monocytes/macrophages. Previously, IL-10 has been reported to suppress HIV-1 replication in monocytes/macrophages at early stages of infection, although virus entry does not appear to be affected (Naif et al., 1996; Ancuta et al., 2001; Kootstra et al., 1994; Wang et al., 1998). However, mechanisms of suppression by IL10 have not yet been elucidated. In this study, we used primary human peripheral blood MDMs to investigate the effects of IL-10 on cyclin T1
Fig. 1. IL-10 inhibits cyclin T1 expression during MDM differentiation. (A) Flow cytometric analysis of cell surface markers. Day 0 monocytes and day 4 macrophages were stained with anti-CD14 or anti-CD71 antibodies (open area). Unstained cells were used as controls (shaded area). (B) 20 μg of total cell lysates prepared at indicated time points were examined by SDS-PAGE followed with immunoblot using the indicated antisera. β-Actin was used as a loading control. (C) Cells were lysed at 6, 14, and 24 h after the addition of IL-10 and were analyzed for expression of the indicated proteins by immunoblotting. For all experiments, a final concentration of 10 ng/ml of IL-10 was used.
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expression and HIV-1 replication. We found that IL-10 downregulates cellular levels of cyclin T1 protein through the induction of proteasome-mediated proteolysis. Furthermore, we found that IL-10 suppresses HIV-1 gene expression in infected MDMs by inhibiting Tat function. Results IL-10 represses cyclin T1 protein expression in differentiating macrophages To examine the effects of IL-10 on cyclin T1 expression, MDMs were differentiated from monocytes using GM-CSF. Flow cytometric analysis showed decreased expression of monocytic marker CD14 and increased expression of macrophage marker CD71 in cells that were not treated with IL-10, indicating the expected differentiation of MDMs (Fig. 1A). MDM cultures treated from day 2 of in vitro differentiation with 10 ng/ml of IL-10 in addition to GM-CSF demonstrated sustained expression of CD14 on day 4. The induction of CD71 surface expression on these cells is similar to that of the MDMs differentiated without IL-10, suggesting that MDMs undergo similar maturation in the presence of IL10 (Fig. 1A). Interestingly, cyclin T1 protein expression was greatly reduced after cells were differentiated in the presence of IL-10 for 2 days (Fig. 1B, compare lanes 2 and 3), while the expression of the 42-kDa isoform of Cdk9, which forms a P-TEFb complex together with cyclin T1, was largely unaffected at this time. To further characterize the kinetics of this down-regulation, cell lysates were prepared at 6, 14, or
Fig. 3. IL-10 down-regulates cyclin T1 expression in macrophages by inducing proteasome-mediated proteolysis. The proteasome inhibitor MG101 (50 nM) was added to IL-10-treated macrophages for 1 h before cells were lysed. 20 μg of total lysates was separated by SDS-PAGE. Levels of indicated proteins were examined by immunoblotting. Data from two independent experiments are presented here.
24 h after the addition of IL-10 on day 2, and the expression levels of cyclin T1 were examined by immunoblot (Fig. 1C). Down-regulation of cyclin T1 was not observed at 6 h after IL-10 addition. It became apparent by 14 h and by 24 h that cyclin T1 was nearly undetectable. These results indicate that the effect of IL-10 on cyclin T1 is a relatively slow process and may be a late event induced by the cytokine. We observed similar kinetics of cyclin T1 down-regulation by IL10 in MDMs from a number of other blood donors. We therefore conclude that IL-10 down-regulates cyclin T1 expression in human macrophages by a relatively slow process, which is likely to be a downstream effect of IL-10 binding to its receptor. IL-10 does not affect cyclin T1 RNA expression
Fig. 2. IL-10 does not affect cyclin T1 RNA expression. Total RNA was isolated from macrophages treated with or without 10 ng/ml of IL-10 at the indicated time points. RNAs were examined by real-time RT-PCR as previously described (Haaland et al., 2005) for the detection of cyclin T1 and CD11c cDNAs. Abundance of the transcripts was normalized to that of GAPDH. Inhibition of cyclin T1 by IL-10 at the protein level was confirmed by immunoblot (see Fig. 1B).
To explore the underlying mechanisms of cyclin T1 downregulation by IL-10, we examined cyclin T1 mRNA levels by quantitative real-time RT-PCR (Fig. 2). As we previously reported using cells differentiated with GM-CSF alone (Liou et al., 2002), cyclin T1 mRNA levels are significantly higher in monocytes than MDMs differentiated for 4 or 7 days. This contrasts to the near absence of cyclin T1 protein expression in day 0 monocytes, and the high-level protein expression in day 4 MDMs (Fig. 1B). Although IL-10 was able to dramatically decrease the level of cyclin T1 protein in day 4 MDMs (Fig. 1B), the amount of cyclin T1 mRNA remained similar in MDMs treated with and without IL-10 (Fig. 2). As a control to validate our real-time RT-PCR assay, RNA levels of CD11c were also examined. CD11c is an integrin whose protein expression is known to be up-regulated through a transcriptional mechanism (Noti and Reinemann, 1995). As expected, CD11c mRNA was strongly induced from day 0 to day 4 in cells treated with GM-CSF alone, while the addition of IL-10 had only a small negative effect on cyclin T1 mRNA on day 4. We therefore conclude that the down-regulation of cyclin T1 expression by IL-10 is not the result of decreased levels of cyclin T1 mRNA.
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ated protein degradation. This process is accelerated by IL-10, and it appears to be the major mechanism involved in the inhibition of cyclin T1 expression by IL-10. IL-10 inhibits cyclin T1 expression in HIV-1-infected macrophages
Fig. 4. IL-10 inhibits cyclin T1 protein expression in HIV-1-infected macrophages. MDMs were infected on day 2 with NL4-3-Luc virus. 10 ng/ml of IL-10 was added to culture on day 3. Cell lysates were prepared on indicated time points and examined by immunoblot.
IL-10 down-regulates cyclin T1 expression through proteasome-mediated proteolysis Because MDMs differentiated with GM-CSF down-regulate cyclin T1 expression at a late differentiation stage by a proteasome-mediated process (Liou et al., 2002), we were interested in examining whether IL-10 activates the same process. We blocked proteasome-mediated proteolysis in IL-10treated macrophages with the proteasome inhibitor, MG101. As shown in Fig. 3 for MDMs from 2 donors, treatment with MG101 for 1 h prior to preparation of cell extracts was able to overcome the suppressive effect of IL-10 on cyclin T1 expression. Treatment with MG101 had no significant effect on the expression of CD11c, the 42-kDa Cdk9, or β-actin, indicating the specificity of its effect on cyclin T1. These results, together with our previous findings (Liou et al., 2002, 2004), indicate that the down-regulation of cyclin T1 protein in differentiating macrophages is mediated by proteasome-medi-
Our previous studies have demonstrated induction of cyclin T1 protein expression by HIV-1 infection (Liou et al., 2004). We therefore examined whether this induction can be overridden by the effect of IL-10. MDMs were infected on day 2 with NL4-3Luc, a reporter virus that encodes the full-length HIV-1 proviral DNA and expresses luciferase enzyme in the place of Nef (Connor et al., 1995). Consistent with our previous findings, cyclin T1 protein expression was shut-off at day 5 in uninfected cultures, whereas it was sustained in the infected culture that was not treated with IL-10 (Fig. 4). In contrast, cyclin T1 expression was absent at both days 4 and 5 in infected cultures that were treated with IL-10. These results indicate that under these conditions, the repression of cyclin T1 expression by IL10 is dominant over the induction by HIV-1 infection. Suppression of cyclin T1 expression by IL-10 contributes to its inhibition on HIV-1 replication in human macrophages Previous studies have indicated that IL-10 specifically inhibits HIV-1 replication in primary human MDMs at a post-entry stage (Ancuta et al., 2001; Kootstra et al., 1994; Wang et al., 1998; Naif et al., 1996). We next examined whether the down-regulation of cyclin T1 by IL-10 contributes to the inhibition of HIV-1 replication in MDMs. To investigate the impact of IL-10 on Tat-mediated
Fig. 5. IL-10 inhibits HIV-1 gene expression in macrophages by suppressing Tat function. (A) 293T cells were infected with either NL4-3-Luc or NL4-3-Luc-Tat− virus. Expression of wild-type Tat protein or mutant Tat-pro18IS was achieved by transfection (empty expression vector was used as a control). Cell lysates were prepared 48 h post-transfection and analyzed for luciferase activity. (B) Differentiating MDMs were infected in triplicate on day 1 with either NL4-3-Luc or NL4-3Luc-Tat− by spinoculation (O'Doherty et al., 2000b). IL-10 (10 ng/ml) was added into culture media on day 3. Cells were harvested every day beginning at day 5. Suppression of cyclin T1 expression in IL-10-treated macrophages was confirmed by immunoblotting (data not shown). Luciferase activities were examined as described above.
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transcription, we constructed a mutant HIV-1 reporter virus, NL4-3Luc-Tat− that lacks a functional Tat protein. This Tat mutant, Tatpro18IS, has been shown previously by plasmid transfection assays to abolish Tat transactivation (Rice and Carlotti, 1990). In infections using 293T cells, the wild-type reporter virus, NL4-3-Luc, expresses efficiently, while mutation of Tat in the NL4-3-LucTat− virus reduced viral expression by ∼32-fold (Fig. 5A). Luciferase expression of the NL4-3-Luc-Tat− virus could be restored to a similar level as that of the wild-type virus by cotransfecting a plasmid expressing the wild-type Tat but not a plasmid expressing the mutant Tat-pro18IS. These results indicate that NL4-3-Luc-Tat− is defective in Tat function, yet it can be fully complemented by exogenously expressing wild-type Tat. We then tested the effects of IL-10 on MDMs infected with these two viruses. MDMs were infected with either NL4-3-Luc or NL4-3Luc-Tat− on day 1 and treated with IL-10 on day 3. From day 5 to day 7, luciferase expression in NL4-3-Luc-infected MDMs was less in IL-10-treated cells than that in untreated cells (Fig. 5B). In contrast, luciferase activities in MDMs infected with the mutant virus, NL4-3-Luc-Tat-, were similar in cells treated with or without IL-10, although the overall expression level in the Tat− virusinfected cells was ∼30-fold lower than the wild-type virus due to the absence of Tat transactivation (Fig. 5B). We observed similar results in infections with these viruses in MDMs from several blood donors. These results indicate that the inhibition of Tat transactivation by IL-10 contributes to the suppression of HIV-1 gene expression by this cytokine. Discussion Despite the well-documented anti-inflammatory functions of IL10, the mechanisms through which IL-10 suppresses macrophage activation are diverse and poorly understood. Phosphorylation and activation of the Jak/Stat pathway are thus far the best-characterized signaling event triggered by IL-10 in macrophages. However, previous studies indicate that one or more additional pathways must also be activated to achieve the inhibitory effect of IL-10 on macrophage activation (Moore et al., 2001). The results presented here show that the IL-10 is able to down-regulate the expression of cyclin T1, a major component of the P-TEFb transcription elongation complex in human MDMs. The suppression of cyclin T1 by IL-10 appears to be due to the induction of proteasomemediated proteolysis, which, to our knowledge, is a novel observation on cellular events triggered by IL-10. It is not clear, however, what signal(s) causes the induction of degradation in MDMs differentiated with GM-CSF in the absence of IL-10. We believe that it is unlikely that the accumulation of IL-10 in the culture medium triggers this event, as we have been unable to detect any activity in the conditioned culture medium from day 7 MDMs, where cyclin T1 had been expressed at low levels, which accelerated the down-regulation of cyclin T1 in early MDM cultures (unpublished results). We also attempted to block the effect of IL-10 by neutralizing it with monoclonal antibodies. We were able to block the down-regulation of cyclin T1 by mixing exogenous IL-10 with antibodies before cytokine addition to cells. However, we have been unable to observe any effect on cyclin T1 expression after the addition of antibodies against any
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endogenous IL-10 which may be secreted into culture media during macrophage differentiation (unpublished data). If IL-10 is involved in the natural shut-off of cyclin T1 expression that occurs late in differentiation in vitro, it is possible that its effect is carried out through autocrine functions rather than paracrine signaling. Proteasome-mediated protein degradation has been identified as a regulatory mechanism for many cellular processes (Pickart, 2004; Ciechanover, 1994). Proteins that undergo rapid turnover usually contain sequence identifiers for targeted degradation, such as PEST regions, KEFRQ motifs and cyclin destruction boxes (Rechsteiner and Rogers, 1996). Cyclin T1 contains a putative PEST sequence in its carboxyl terminus (Wei et al., 1998), which may specify cyclin T1 degradation by the ubiquitylation and proteasome pathway. Alternatively, it is possible that an unidentified cellular factor is required for the translation of cyclin T1 mRNA, and this positive factor may be down-regulated through IL-10-induced proteolysis; blockage of proteasome-mediated proteolysis by MG101 may increase the level of this putative positive factor, and this would result in increased translation of cyclin T1 mRNA and a subsequent increase in the level of cyclin T1 protein. Our data cannot distinguish these possibilities, although we favor the former because it is commonly involved in regulating levels of cellular transcription factors and cyclin-cdk complexes (Obaya and Sedivy, 2002; Muratani and Tansey, 2003). Interestingly, suppressing cyclin T1 expression through proteasome-mediated degradation appears to be specific for macrophages as treatment of peripheral blood lymphocytes, Jurkat, and 293T cells with proteasome inhibitors does not affect its protein level (Liou et al., 2004). Transcription profiling studies in control MDMs and MDMs treated with IL-10 are currently underway in an attempt to identify enzymes and accessory proteins induced by IL-10 that may be involved in the down-regulation of cyclin T1. To date, reports on the impact of IL-10 on HIV-1 replication in monocytes/macrophages have been divergent and dependent on the cell types and amounts of cytokine used in the experiments (Stearns et al., 2003; Sozzani et al., 1998; Ancuta et al., 2001; Kootstra et al., 1994; Naif et al., 1996; Wang et al., 1998). Our data are also consistent with the findings that IL-10, by itself, affects HIV-1 replication negatively in macrophages. In conclusion, our results demonstrate that IL-10 downregulates the expression of cyclin T1, probably though the activation of proteasome-mediated proteolysis of this protein. We also present evidence that the inhibition of HIV-1 replication in MDMs by IL-10 involves the suppression of Tat transactivation function during the transcription of HIV-1 provirus. Our findings, together with other reports (Moore et al., 2001), suggest that IL-10 may affect multiple stages of the HIV-1 life cycle, and its induced expression in progressive AIDS patients may have a complex effect on HIV-1 pathogenesis. Materials and methods Reagents and antibodies Human granulocyte–macrophage colony-stimulating factor (GM-CSF) and interleukin-10 (IL-10) were purchased from R&D
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Systems. Anti-CD14-PE and anti-CD71-FITC were from BD PharMingen. Primary antibodies against cyclin T1 (T-18), Cdk9 (C-20) and CD11c (C-20) were purchased from Santa Cruz Biotechnology. Anti-β-actin antibody and MG101 were from Sigma. Isolation and differentiation of monocytes/macrophages Peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coat of healthy blood donors (Gulf Coast Regional Blood Center) by Ficoll-Hypaque density gradient centrifugation (Isolymph [Gallard/Schlesinger]). Monocytes were isolated by an adherence method in which 3 × 107 PBMCs were seeded onto each 10-cm plastic tissue culture dish (Sarstedt) containing RPMI-1640 (Invitrogen) supplemented with 1% human serum (Sigma) and 1% antibiotics (Invitrogen). Following incubation at 37 °C for 1 h, non-adherent cells were removed, and adherent monocytes were washed vigorously with pre-warmed phosphate-buffered saline (PBS). Cells were further purified by incubating at 37 °C for 2 h in complete medium (RPMI-1640 + 10% fetal bovine serum + antibiotics) and washing with pre-warmed PBS vigorously for 3 times. Monocytes were then cultured in complete medium containing 10 U/ml GM-CSF to allow differentiation into macrophages. For IL-10 treatment, a one-time addition of IL-10 was introduced on day 2 to a final concentration of 10 ng/ml. Initial seeding conditions for other formats of tissue culture vessels are 2 × 106 PBMCs per 6-cm dish or 1 × 106 PBMCs per well in a 6-well plate. In some experiments, monocytes were also isolated from PBMCs using the negative selection-based Monocyte Isolation Kit II (Miltenyi Biotech.) to achieve a purity of 90% or higher as determined by flow cytometry.
by measuring OD260 nm of the sample. 1 μg of the total RNA was reverse transcribed using iScript cDNA synthesis kit (BioRad) in a 20 μl reaction according to the manufacturer's protocol. Products from these reactions were diluted 1:10 and used as templates in 15 μl real-time PCR reactions containing 1/ 2 volume of 2× iQ SYBR Green Supermix (Bio-Rad) and 200 nM final concentration of the appropriate primers. Triplicate PCR reactions were run in 96-well format using a Bio-Rad iCycler. After the initial hot start at 95 °C for 3 min, 40 cycles of amplification were carried out for 15 s at 95 °C followed by 1-min annealing/extension at 55 °C (data were collected at this step). Analysis was performed using the MyIQ software program (Bio-Rad), which automatically determines the threshold crossing (CT) value for each reaction. A five-fold dilution series of HeLa cell total RNA was used to determine the amplification efficiency (E) of each primer pair. The relative expression ratio (R) of a gene of interest (g.o.i., normalized to day 0) was calculated with the following formula using GAPDH as a reference gene (Pfaffl, 2001). DCTg:o:i:ðday 0sampleÞ 1 þ Eg:o:i: R¼ ð1 þ Eref ÞDCTref ðday 0sampleÞ Sequences of the primers used here are as follows: cyclin T1 (sense 5′-AACCTTCGCCGCTGCCTTC-3′; anti-sense 5′ACC GTTTGTTGTTGTTCTTCCTCTC-3′), CD11c (sense 5′-TTTGAGTGTCGGGAGCAGGTG-3′; anti-sense 5′GCCAGGTCCAAGGTCACAGAG-3′), GAPDH (sense 5′CGCCAGCCGAGCCACATC-3′; anti-sense 5′-AATCCCTTGACTCCGACCTTCA-3′). Virus production and infections
Flow cytometry Adherent cells were collected by incubating the dishes at 4 °C with PBS containing 2% FBS and 2 mM EDTA for 10 min and detaching the cells using a rubber scraper. Cells were washed twice with cold PBS containing 2% FBS and stained for 15 min at 4 °C with appropriate antibodies. After washing, cells were fixed in 1% paraformaldehyde (Fisher) and analyzed using a Coulter XL-MCL flow cytometer. Immunoblots Cell lysates were prepared using Luciferase Cell Lysis Buffer (Promega) according to the manufacturer's protocol. Protein concentration was determined by a Bradford assay (Bio-Rad). 20 μg of total lysate was separated by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). The procedure for immunoblotting using SuperSignal West Pico Chemiluminescent Substrate (Pierce) was described previously (Herrmann et al., 1996). Isolation of total RNA and analysis by quantitative real-time RT-PCR Total RNA was extracted from frozen cell pellets using the RNeasy mini kit (Qiagen). RNA concentration was determined
pNL-Luc-E−R+ was obtained from the NIH AIDS Research and Reference Reagent Program and has been described previously (Connor et al., 1995). pNL-Luc-E−R+ encodes for full-length NL4-3 HIV-1 proviral DNA with a frameshift in Env and expresses luciferase enzyme in place of Nef. To generate the Tat-null virus, NL4-3-LucTat−, an EcoRI restriction endonuclease site (GAATTC) was inserted between residues 18 and 19 within the Tat-encoding sequence of pNL-Luc-E−R+ using QuickChange II XL site-directed mutagenesis kit (Stratagene). For virus production, 5 × 106 293T cells were transfected with 10 μg of either pNL-Luc-E-R+ or pNL-Luc-E−R+Tat−, along with 10 μg of plasmids expressing VSV-G protein (pseudotyped envelope) and HIV-1 Tat protein by calcium phosphate transfection (Sambrook and Russell, 2001). Culture supernatant containing infectious virions was harvested 72 h posttransfection and cleared of cell debris by centrifuging at 1000×g for 10 min. Virus concentration was determined by the COULTER HIV-1 p24 antigen assay using protocols supplied by the manufacturer. Virus stock was stored at −80 °C in working aliquots. For infection of 293T cells, 3 × 105 293T cells (ATCC) in 12-well plates were infected in triplicate with 5 μg of either NL4-3-Luc or NL4-3-Luc-Tat−. Infection of primary human MDMs was carried out in 6-well plates using 50 μg/ml of either virus. The method of spinoculation was employed to
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achieve efficient infection. As previously described (O'Doherty et al., 2000a), cells were incubated with virus-containing media under 2000×g of centrifugational force for 2 h. Unbound virus was removed by washing the cells 3 times with cold PBS. Complete media containing 10 U/ml GM-CSF were added to the cells for further culturing. Transfections and luciferase assay Transient transfection was carried out using Lipofectamine 2000 transfection reagent (Invitrogen). 3 × 105 293T cells, infected with either NL4-3-Luc or NL4-3-Luc-Tat− , were transfected in triplicates with 0.5 μg of either the control vector, Tat-expressing or Tat mutant-expressing plasmid. Cells were harvested 48 h later. Luciferase activity in cell lysates was detected using the Luciferase Assay System from Promega according to protocols supplied therein. Acknowledgments We thank Dr. Ned Landau for the kind gift of plasmid pNL43.Luc.R+.E−. We also thank Dr. Chris Herrmann for comments on the manuscript and Dr. Li-Ying Liou for useful discussions and technical advice. This work was supported by NIH grant AI45374. References Ancuta, P., Bakri, Y., Chomont, N., Hocini, H., Gabuzda, D., HaeffnerCavaillon, N., 2001. Opposite effects of IL-10 on the ability of dendritic cells and macrophages to replicate primary CXCR4-dependent HIV-1 strains. J. Immunol. 166, 4244–4253. Barboric, M., Peterlin, B.M., 2005. A new paradigm in eukaryotic biology: HIV Tat and the control of transcriptional elongation. PLoS Biol. 3, e76. Ciechanover, A., 1994. The ubiquitin-proteasome proteolytic pathway. Cell 79, 13–21. Clerici, M., Shearer, G.M., 1993. A TH1→TH2 switch is a critical step in the etiology of HIV infection. Immunol. Today 14, 107–111. Connor, R.I., Chen, B.K., Choe, S., Landau, N.R., 1995. Vpr is required for efficient replication of human immunodeficiency virus type-1 in mononuclear phagocytes. Virology 206, 935–944. Fujinaga, K., Irwin, D., Huang, Y., Taube, R., Kurosu, T., Peterlin, B.M., 2004. Dynamics of human immunodeficiency virus transcription: P-TEFb phosphorylates RD and dissociates negative effectors from the transactivation response element. Mol. Cell. Biol. 24, 787–795. Garber, M.E., Jones, K.A., 1999. HIV-1 Tat: coping with negative elongation factors. Curr. Opin. Immunol. 11, 460–465. Haaland, R.E., Yu, W., Rice, A.P., 2005. Identification of LKLF-regulated genes in quiescent CD4+ T lymphocytes. Mol. Immunol. 42, 627–641. Herrmann, C.H., Gold, M.O., Rice, A.P., 1996. Viral transactivators specifically target distinct cellular protein kinases that phosphorylate the RNA polymerase II C-terminal domain. Nucleic Acids Res. 24, 501–508. Ivanov, D., Kwak, Y.T., Guo, J., Gaynor, R.B., 2000. Domains in the SPT5 protein that modulate its transcriptional regulatory properties. Mol. Cell. Biol. 20, 2970–2983. Karn, J., 1999. Tackling Tat. J. Mol. Biol. 293, 235–254. Kedzierska, K., Crowe, S.M., 2002. The role of monocytes and macrophages in the pathogenesis of HIV-1 infection. Curr. Med. Chem. 9, 1893–1903. Kedzierska, K., Crowe, S.M., Turville, S., Cunningham, A.L., 2003. The influence of cytokines, chemokines and their receptors on HIV-1
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